AOP 10: Binding to the picrotoxin site of ionotropic GABA ......

Organisation for Economic Co-operation and Development

DOCUMENT CODE

For Official Use English - Or. English

1 January 1990

AOP 10: Binding to the picrotoxin site of ionotropic GABA receptors leading to

epileptic seizures in adult brain

Short Title: Blocking iGABA receptor ion channel leading to seizures

This document was approved by the Extended Advisory Group on Molecular Screening and

Toxicogenomics in June 2018.

The Working Group of the National Coordinators of the Test Guidelines Programme and the

Working Party on Hazard Assessment are invited to review and endorse the AOP by 29 March

2019.

Magdalini Sachana, Administrator, Hazard Assessment, [email protected], +(33-

1) 85 55 64 23

Nathalie Delrue, Administrator, Test Guidelines, [email protected], +(33-1) 45 24 98

44

This document, as well as any data and map included herein, are without prejudice to the status of or sovereignty over any territory, to the

delimitation of international frontiers and boundaries and to the name of any territory, city or area.

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Foreword

This Adverse Outcome Pathway (AOP) on Binding to the picrotoxin site of ionotropic

GABA receptors leading to epileptic seizures in adult brain, has been developed under the

auspices of the OECD AOP Development Programme, overseen by the Extended Advisory

Group on Molecular Screening and Toxicogenomics (EAGMST), which is an advisory

group under the Working Group of the National Coordinators for the Test Guidelines

Programme (WNT). The AOP has been reviewed internally by the EAGMST, externally

by experts nominated by the WNT, and has been endorsed by the WNT and the Working

Party on Hazard Assessment (WPHA) in xxx.

Through endorsement of this AOP, the WNT and the WPHA express confidence in the

scientific review process that the AOP has undergone and accept the recommendation of

the EAGMST that the AOP be disseminated publicly. Endorsement does not necessarily

indicate that the AOP is now considered a tool for direct regulatory application.

The Joint Meeting of the Chemicals Committee and the Working Party on Chemicals,

Pesticides and Biotechnology agreed to declassification of this AOP on xxx.

This document is being published under the responsibility of the Joint Meeting of the

Chemicals Committee and the Working Party on Chemicals, Pesticides and Biotechnology.

The outcome of the internal and external reviews are publicly available respectively in the

AOP Wiki and the eAOP Portal of the AOP Knowledge Base at the following links:

[internal review] [external review].

https://aopwiki.org/

http://aopkb.org/

http://www.oecd.org/chemicalsafety/testing/internal-review-charge-questions-aop-10.pdf

https://aopkb.oecd.org/DocumentControler.axd?e=%c2%a3%c2%a41a8V71E60xbrdsd5R4KLMQ%3d%3d%c2%b5%c2%a4GBKP4eqw5wAGKqIH67JNeOqFfZzjfBxx1yzYiPrjVPHYVyWSxhEPUcblalYeZleFGMliHqldP6v0hIOWyh6Xsw%3d%3d&a=g&id=5043158a-9c3d-4a7a-8fd9-63008827291c

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Table of contents

Foreword ................................................................................................................................................ 2

Adverse Outcome Pathway on Binding to the picrotoxin site of ionotropic GABA receptors

leading to epileptic seizures in adult brain .......................................................................................... 5

Short Title: Blocking iGABA receptor ion channel leading to seizures .............................................. 5 Authors: ............................................................................................................................................ 5

Abstract .................................................................................................................................................. 6

Background ............................................................................................................................................ 7

Graphical Representation ..................................................................................................................... 8

Summary of the AOP ............................................................................................................................ 9

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO) ............................... 9 Key Event Relationships ...................................................................................................................... 9 Stressors ............................................................................................................................................. 10

Overall Assessment of the AOP .......................................................................................................... 12

Biological plausibility ........................................................................................................................ 12 Concordance of dose-response relationships ..................................................................................... 12 Temporal concordance among the key events and the adverse outcome ........................................... 12 Strength, consistency, and specificity of association of adverse effect and initiating event .............. 13 Uncertainties, inconsistencies, and data gaps .................................................................................... 13

Domain of Applicability ...................................................................................................................... 14

Life Stage Applicability ..................................................................................................................... 14 Taxonomic Applicability ................................................................................................................... 14 Sex Applicability ............................................................................................................................... 14

Essentiality of the Key Events ............................................................................................................ 15

Weight of Evidence Summary ............................................................................................................ 15

Quantitative Consideration ................................................................................................................ 16

Considerations for Potential Applications of the AOP (optional) ................................................... 17

References ............................................................................................................................................ 17

Appendix 1 ........................................................................................................................................... 20

List of MIEs in this AOP ................................................................................................................... 20 Event: 667: Binding at picrotoxin site, iGABAR chloride channel ............................................... 20

List of Key Events in the AOP .......................................................................................................... 26 Event: 64: Reduction, Ionotropic GABA receptor chloride channel conductance ......................... 26 Event: 669: Reduction, Neuronal synaptic inhibition .................................................................... 29 Event: 682: Generation, Amplified excitatory postsynaptic potential (EPSP) ............................... 31 Event: 616: Occurrence, A paroxysmal depolarizing shift ............................................................ 34

List of Adverse Outcomes in this AOP .............................................................................................. 37 Event: 613: Occurrence, Epileptic seizure ..................................................................................... 37

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Appendix 2 ........................................................................................................................................... 41

List of Adjacent Key Event Relationships ......................................................................................... 41 Relationship: 666: Binding at picrotoxin site, iGABAR chloride channel leads to Reduction,

Ionotropic GABA receptor chloride channel conductance ............................................................ 41 Relationship: 667: Reduction, Ionotropic GABA receptor chloride channel conductance leads

to Reduction, Neuronal synaptic inhibition .................................................................................... 44 Relationship: 683: Reduction, Neuronal synaptic inhibition leads to Generation, Amplified

excitatory postsynaptic potential (EPSP) ....................................................................................... 47 Relationship: 684: Generation, Amplified excitatory postsynaptic potential (EPSP) leads to

Occurrence, A paroxysmal depolarizing shift ................................................................................ 52 Relationship: 630: Occurrence, A paroxysmal depolarizing shift leads to Occurrence, Epileptic

seizure ............................................................................................................................................ 55

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Adverse Outcome Pathway on Binding to the picrotoxin site

of ionotropic GABA receptors leading to epileptic seizures in

adult brain

Short Title: Blocking iGABA receptor ion channel leading to seizures

Authors:

Ping Gong, Edward J. Perkins, US Army Engineer Research and Development Center

Email: [email protected] or [email protected]

Point of contact for this AOP entry: Dr. Ping Gong

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Abstract

This AOP begins with a molecular initiating event (MIE) where a chemicla binds to the

picrotoxin binding site at or near the central pore of the ionotropic GABA receptor complex

causing blockage of the ion channel. As a result, the first key event (KE) is a decrease in

inward chloride conductance through the ligand-gated ion channel. This leads to the second

KE, a reduction in postsynaptic inhibition, reflected as reduced frequency and amplitude

of spontaneous inhibitory postsynaptic current (sIPSC) or abolishment of GABA-induced

firing action in GABAergic neuronal membranes. Consequently, the resistance of

excitatory neurons to fire is decreased, resulting in the generation of a large excitatory

postsynaptic potential (EPSP), i.e., the third KE. The large EPSP is reflected as a spike

(rise) of intracellular Ca2+ observed in the affected region, where a large group of

excitatory neurons begin firing in an abnormal, excessive, and synchronized manner. Such

a giant Ca2+-mediated excitatory firing (depolarization) causes voltage-gated Na+ to open,

which results in action potentials. The depolarization is followed by a period of hyper-

polarization mediated by Ca2+-dependent K+ channels or GABA-activated Cl− influx.

During seizure development, the post-depolarlization hyperpolarization becomes smaller,

gradually disappears, and is replaced by a depolarization. This characteristic

depolarization-shrinking hyperpolarization sequence of events represents the fourth KE

known as “paroxysmal depolarizing shift” (PDS), which forms a “seizure focus”. A PDS

is, essentially, an indication of epilepsy at the cellular level, which serves as the foci to

initiate the adverse outcome at the organismal level of epileptic seizure. The severity of

symptoms is often dose- and duration- dependent, while the toxicological symptoms are

associated with the type and location of affected iGABARs. Mortality can occur if the

individual sustains a prolonged or pronounced convulsion or seizure. Neurotoxicity, of

which seizures are an end point, is a regulated outcome for chemicals. This AOP allows for

screening chemicals for the potential to cause neurotoxicity through the use of in vitro

assays that demonstrate binding to the picrotoxin site, electrophysiological assays

demonstrating depolarization of neuronal membranes, or electroencephalography that

records electrical activity of the adult brain.

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Background

Ionotropic GABA receptors (iGABARs) are ligand-gated ion channels which play

important functional roles in the nervous system. As the major player in inhibitory

neurotransmission, iGABARs are widely distributed in both vertebrates and invertebrates

(McGonigle and Lummis 2010; Garcia-Reyero et al. 2011). In vertebrates, the iGABAR

includes two subclasses of fast-responding ion channels, GABAA receptor (GABAA-R)

and GABAC receptor (GABAC-R). Invertebrate iGABARs do not readily fit the vertebrate

GABAA/GABAC receptor categories (Sieghart 1995). The majority of insect iGABARs

are distinguished from vertebrate GABAA receptors by their insensitivity to bicuculline

and differ from GABAC-Rs in that they are subject to allosteric modulation, albeit weakly,

by benzodiazepines and barbiturates (Hosie et al. 1997).

Chemical interactions with iGABARs can cause a variety of pharmacological and

neurotoxicological effects depending on the location of the active or allosteric site affected.

Three distinct types of interactions at binding sites on iGABARs can antagonize the

postsynaptic inhibitory functions of GABA and lead to epileptic seizures and death. These

three types of interactions correspond to three AOPs (Gong et al. 2015). One of the three

types of interaction is non-competitive channel blocking at the picrotoxin convulsant site

located inside of the iGABAR pore that spans neuronal cell membranes (this MIE). The

other two types of interactions are negative modulation at allosteric sites and competitive

binding at the active orthosteric sites (MIEs to be developed in the future).

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Graphical Representation

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Summary of the AOP

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes

(AO)

Sequence Type Event

ID Title Short name

1 MIE 667 Binding at picrotoxin site,

iGABAR chloride channel

Binding at picrotoxin site,

iGABAR chloride channel

2 KE 64

Reduction, Ionotropic

GABA receptor chloride

channel conductance

Reduction, Ionotropic

GABA receptor chloride

channel conductance

3 KE 669 Reduction, Neuronal

synaptic inhibition

Reduction, Neuronal

synaptic inhibition

4 KE 682

Generation, Amplified

excitatory postsynaptic

potential (EPSP)

Generation, Amplified

excitatory postsynaptic

potential (EPSP)

5 KE 616 Occurrence, A paroxysmal

depolarizing shift

Occurrence, A paroxysmal

depolarizing shift

6 AO 613 Occurrence, Epileptic

seizure

Occurrence, Epileptic

seizure

Key Event Relationships

Upstream Event Relationship

Type

Downstream

Event Evidence

Quantitative

Understanding

Binding at

picrotoxin site,

iGABAR

chloride channel

adjacent

Reduction,

Ionotropic GABA

receptor chloride

channel

conductance

High High

Reduction,

Ionotropic

GABA receptor

chloride channel

conductance

adjacent

Reduction,

Neuronal synaptic

inhibition

High High

Reduction,

Neuronal

synaptic

inhibition

adjacent

Generation,

Amplified

excitatory

postsynaptic

potential (EPSP)

High Moderate

Generation,

Amplified

excitatory

adjacent

Occurrence, A

paroxysmal

depolarizing shift

Moderate Moderate

https://aopwiki.org/events/667














https://aopwiki.org/relationships/666
















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postsynaptic

potential (EPSP)

Occurrence, A

paroxysmal

depolarizing shift

adjacent Occurrence,

Epileptic seizure High Moderate

Stressors

Name Evidence

Picrotoxin High

Lindane High

Dieldrin High

Heptachlor High

Endosulfan High

RDX High

Fipronil High

1. Picrotoxin

Picrotoxin seizures are well defined mechanistically. They arise from GABAA receptor

chloride channel blockage. (See Page 131 in "Models of Seizures and Epilepsy", edited by

A. Pitkanen, P.A. Schwartzkroin, S.L. Moshe. Elsevier Academic Press. 2006)

As picrotoxin effectively inhibits chloride influx in GABAA and other ionotropic receptors,

it represents a universal "reference" channel blocker with whom other ligands may be

compared. (A.V. Kalueff. 2007. Mapping convulsants' binding to the GABA-A receptor

chloride ionophore: a proposed model for channel binding sites. Neurochem Int. 50(1): 61-

8.)

2. Lindane

Neurotoxic pesticides, such as lindane, alpha-endosulfan and dieldrin, share structural

similarities (and compete for the binding site) with picrotoxin, inhibit TBPS binding,

induce seizures and block Cl-currents through the ionophore. (A.V. Kalueff. 2007.

Mapping convulsants' binding to the GABA-A receptor chloride ionophore: a proposed

model for channel binding sites. Neurochem Int. 50(1): 61-8.)

3. Dieldrin

See evidence text for picrotoxin and lindane.

4. Heptachlor

Organochlorine insecticides, including DDT, dieldrin, heptachlor, lindane, and β-

hexachlorocyclohexane (β-HCH), have varied targets and mechanisms of action within the

central nervous system that affect the function of sodium and calcium channels and

transporters. They also interfer with γ-aminobutyric acid (GABA) neurotransmission by

blocking specific GABA receptors, contributing to their neurotoxic effects (Narahashi et

al., 1995).

5. Endosulfan

See "Toxicological Profile for Endosulfan" (Agency for Toxic Substances and Disease

Registry, 2000). Dose- and time-dependent correlations between picrotoxin site binding






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by chlorinated pesticides and convulsions were observed in mice (Cole and Casida 1986),

whereas poisoning with the organochlorine insecticide endosulfan caused seizure, status

epilepticus, or refractory status epilepticus in humans (Durukan et al. 2009;

Moon et al. 2017; Moses and Peter 2010; Parbhu et al. 2009; Roberts et al. 2004), and

eventually led to the death of a farmer (Roberts et al. 2004) and a toddler (Parbhu et al.

2009).

6. RDX

See Williams et al. (2011).

7. Fipronil

See Chen et al. (2006).

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Overall Assessment of the AOP

Biological plausibility

The biological mechanisms underlying epilepsy (defined as a disorder of the central

nervous system characterized by recurrent seizures unprovoked by an acute systemic or

neurologic insult) have been investigated for more than six decades and are well understood

except for a few intermediate details (Bromfield et al. 2006; Lomen-Hoerth and Messing

2010). As one of the cellular mechanisms of action, blocking postsynaptic GABA-mediated

inhibition can lead to epileptic seizure (Dichter and Ayala 1987; Gong et al. 2015). It has

been extensively documented that non-competitive ion channel blockers such as picrotoxin,

lindane, α-endosulfan and fipronil act through binding to iGABARs (Chen et al. 2006;

Casida and Durkin 2015). Despite large structural diversity, it has been postulated that these

blockers fit a single binding site in the chloride channel lumen lined by five TM2

(TransMembrane domain 2) segments, which was supported in the β3 homopentamer by

mutagenesis, pore structure studies, ligand binding, and molecular modeling (Chen et al.

2006; Casida and Durkin 2015). The downstream cascading key events of this AOP have

also been reviewed in multiple publications (e.g., Dichter and Ayala 1987; Bromfield et al.

2006; Lomen-Hoerth and Messing 2010). Based on the extensive evidence supporting the

MIE, KEs and the AO, there is a high likelihood and certainty that such GABA antagonists

as non-competitive channel blockers produce seizures in both invertebrates and vertebrates

that possess GABAergic inhibitory neurotransmission in central nervous systems (Treiman

2001; Raymond-Delpech et al. 2005).

Concordance of dose-response relationships

Numerous pharmacological studies have reported quantitative dose-response relationships

between the dose of non-competitive antagonists and the recorded electrophysirological

esponse of epileptic seizures. See examples for picrotoxin (Newland and Cull-Candy 1992;

Ikeda 1998; Stilwell et al. 2006), RDX (Williams et al. 2011) and dieldrin (Babot et al.

2007; Ikeda 1998).

Temporal concordance among the key events and the adverse outcome

Given that the basic mechanism of neuronal excitability is the action potential, a

hyperexcitable state can result from many causes including decreased inhibitory

neurotransmission (KE2). Moreover, action potentials occur due to depolarization of the

neuronal membrane, with membrane depolarization propagating down the axon to induce

neurotransmitter release at the axon terminal. The action potential occurs in an all-or-none

fashion as a result of local changes in membrane potential brought about by net positive

inward ion fluxes. Membrane potential thus varies with the activation of ligand-gated

channels, whose conductance is affected by binding to neurotransmitters. For instance, the

conductance is decreased (KE1) due to the binding at allosteric sites in the chloride channel

of iGABAR by non-competitive blockers (MIE).

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Seizure initiation: The hypersynchronous discharges that occur during a seizure may begin

in a very discrete region of the cortex and then spread to neighboring regions. Seizure

initiation is characterized by two concurrent events: 1) high-frequency bursts of action

potentials, and 2) hypersynchronization of a neuronal population. The synchronized bursts

from a sufficient number of neurons result in a so-called spike discharge on the EEG

(electroencephalogram), i.e., amplified excitatory postsynaptic potential (KE3). At the

level of single neurons, epileptiform activity consists of sustained neuronal depolarization

resulting in a burst of action potentials, a plateau-like depolarization associated with

completion of the action potential burst, and then a rapid repolarization followed by

hyperpolarization. This sequence is called the paroxysmal depolarizing shift (KE4).

Seizure propagation (AO), the process by which a partial seizure spreads within the brain,

occurs when there is sufficient activation to recruit surrounding neurons. This leads to a

loss of surround inhibition and spread of seizure activity into contiguous areas via local

cortical connections, and to more distant areas via long association pathways such as the

corpus callosum. The propagation of bursting activity is normally prevented by intact

hyperpolarization and a region of surrounding inhibition created by inhibitory neurons.

With sufficient activation there is a recruitment of surrounding neurons via a number of

mechanisms. The above description is excerpted and summarized from Bromfield et al.

(2006).

Strength, consistency, and specificity of association of adverse effect and

initiating event

Drug- or chemical-induced focal or generalized seizures are not limited to any specific

group of chemical structures, neuroreceptors or taxonomy. This AOP addresses a specific

group of chemicals that are capable of binding to the picrotoxin convulsant site of

iGABARs, leading to epileptic seizures. Literature evidence strongly and consistently

supports such a forward association, i.e., binding to the picrotoxin site leads to epileptic

seizures (see reviews Gong et al. 2015; Bromfield et al. 2006; Raymond-Delpech et al.

2005; Treiman 2001; Dichter and Ayala 1987). For instance, dose- and time-dependent

correlations between picrotoxin site binding by chlorinated pesticides and convulsions

were observed in mice (Cole and Casida 1986), whereas poisoning with the organochlorine

insecticide endosulfan caused seizure, status epilepticus, or refractory status epilepticus in

humans (Durukan et al. 2009; Moon et al. 2017; Moses and Peter 2010; Parbhu et al. 2009;

Roberts et al. 2004), and eventually led to the death of a farmer (Roberts et al. 2004) and a

toddler (Parbhu et al. 2009).

Uncertainties, inconsistencies, and data gaps

No inconsistencies have been reported so far, though some uncertainties and data gaps do

exist. For instance, the process by which seizures typically end, usually after seconds or

minutes, and what underlies the failure of this spontaneous seizure termination in the life-

threatening condition known as status epilepticus are less well understood (Bromfield et al.

2006). The spread of epileptic activity throughout the brain, the development of primary

generalized epilepsy, the existence of “gating", mechanisms in specific anatomic locations,

and the extrapolation of hypotheses derived from simple models of focal epilepsy to explain

more complex forms of epilepsies observed in human and other animals, all are also not

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yet fully understood (Dichter and Ayala 1987). The remarkable plasticity of GABAergic

neurons and iGABARs (e.g., iGABAR regulation by phosphorylation, and expression of

potassium chloride cotransporters (KCCs) or sodium dependent anion exchangers

(NDAE)) in response to insults and injury constitutes additional complexity

and creates another layer of uncertainties in the emergence of epileptic seizures (Ben-Ari

2006; Galanopoulou 2008; Scharfman and Brooks-Kayal 2014).

Domain of Applicability

Life Stage Applicability

Life Stage Evidence

Adults High

Taxonomic Applicability

Term Scientific Term Evidence Links

human Homo sapiens High NCBI

mouse Mus musculus High NCBI

rat Rattus norvegicus High NCBI

bobwhite quail Colinus virginianus High NCBI

zebrafish Danio rerio Moderate NCBI

Sex Applicability

Sex Evidence

Male High

Female High

This AOP is applicable to all vertebrates and invertebrates possessing iGABARs, without

restrictions pertaining to sex and taxonomy. This AOP may not be applicable to young

animals during their embryonic and early developmental stages when GABA acts as an

excitatory neurotransmitter due to increased intracellular Clˉ concentration in immature or

developing neurons (Taketo and Yoshioka 2000; Galanopoulou 2008; Ben-Ari 2006). A

key feature of the immature type function of GABAA receptors is the depolarizing signaling,

attributed to the inability of young neurons to maintain low intracellular chloride. The

regulation of GABAergic switch is different in neurons with depolarizing vs

hyperpolarizing GABAergic signaling. In mature neurons, recurrent and prolonged

seizures may trigger a pathological reemergence of immature features of GABAA receptors,

http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606





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which compromises the efficacy of GABA-mediated inhibition. In immature neurons with

depolarizing GABAergic signaling, the physiological and pathological regulation of this

system is completely different, possibly contributing to the different outcomes of early life

seizures (Galanopoulou 2008).

Essentiality of the Key Events

The MIE, four key events and resulted adverse outcome listed for this AOP are all essential

based on current knowledge and understanding of the structure, pharmacology,

localization, classification of ionotropic GABA receptors (e.g., GABAA receptors) (Olsen

2015; Olsen and Sieghart 2009), the basic neurophysiology, neurochemistry and cellular

mechanisms underlying epilepsies (Dichter and Ayala 1987; Bromfield et al. 2006), and

the pathophysiology of seizures (Lomen-Hoerth and Messing 2010).

Weight of Evidence Summary

A novel subject matter expertise driven approach was developed for weight of evidence

(WoE) assessment (Collier et al. 2016). This approach, tailored toward the needs of AOPs

and in compliance with the AOP Users' Handbook (OECD 2017), was based on criteria

and metrics related to data quality and causality (i.e., the strength of causal linkage between

key events). The methodology consists of three main steps: (1) assembling evidence

(preparing the AOP), (2) weighting evidence (criteria weighting and scoring), and (3)

weighting the body of evidence (aggregating lines of evidence). We adopted the General

Assessment Factors (GAF) established by the US EPA as the criteria for data quality

evaluation, and a set of five criteria known as Bradford Hill criteria to measure the strength

of causal linkages (see Table below). The numerical scoring scale corresponds to the

descriptive scoring scale recommended in the Users' Handbook as follows: 4 or 5 is

equivalent to High, 3 is equivalent to Moderate, and 1 or 2 is equivalent to Low. Detailed

description on, supporting evidence for and quantitative understanding of each KE/KER

can be found by clicking the link on each KE/KER above.

The authors of Collier et al. (2016), who served as the developers for several AOPs

(including this one), represented subject matter experts, and they applied their expertise

and best professional judgment to assign weights to the criteria and scores to each line of

evidence. Final criteria scoring represented the consensus scores agreed upon after debates

among the authors. For example, the MIE has been intensively reviewed where numerous

documented studies provided supporting evidence. Hence, the MIE received high scores

for all five GAF criteria. However, the Bradford Hill criteria connecting KE2-->KE3 and

KE3-->KE4 received relatively lower scores because there still exist knowledge gaps in

16 │

the spread of epileptic activity throughout the normal CNS and the mechanism underlying

the generalized epilepsies. The following table shows the results of our WoE assessment

(note that scores may be inexact due to rounding).

Please note that this table and scoring system might not be consistent with the Users’

Handbook.

Quantitative Consideration

Many studies have reported quantitative relationships between chemicals such as drugs and

pesticides and electrophysiological response. For instance, long-term exposure of primary

cerebellar granule cell cultures to 3 µM dieldrin reduced the GABAA receptor function to

55% of control, as measured by the GABA-induced 36Cl- uptake (Babot et al. 2007). Juarez

et al. (2013) observed that picrotoxin exerted concentration-dependent and reversible

inhibition of GABA-induced membrane currents in primary cultured neurons obtained

from the guinea-pig small intestine. The stepwise qualitative relationships between

consecutive events (MIE, KEs and AO) are well established but quantitative ones are rarely

documented.

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Considerations for Potential Applications of the AOP (optional)

This AOP can be used to establish the mode of neurotoxicological actions for chemicals

capable of binding to the picrotoxin convulsant site of iGABARs. It can also be applied to

risk assessment where AOP can assist in predictive modeling of chemical toxicity.

Chemicals acting through this AOP can be distinguished from neurotoxicants acting on

other types of iGABAR sites (e.g., orthosteric or allosteric binding sites) or other types of

neuroreceptors (e.g., ardrenergic, dopaminergic, glutaminergic, cholinergic and

serotonergic receptors). More information relevant to this topic can be found in Gong et al.

(2015).

References

Babot Z, Vilaro M T, Sunol C. (2007) Long-term exposure to dieldrin reduces gamma-

aminobutyric acid type A and N-methyl-D-aspartate receptor function in primary cultures

of mouse cerebellar granule cells. J Neurosci Res, 85(16):3687-3695.

Ben-Ari Y. (2006) Seizures beget seizures: the quest for GABA as a key player. Crit Rev

Neurobiol. 18(1-2):135-44.

Bromfield EB, Cavazos JE, Sirven JI. (2006) Chapter 1, Basic Mechanisms Underlying

Seizures and Epilepsy. In: An Introduction to Epilepsy [Internet]. West Hartford (CT):

American Epilepsy Society; Available from:

http://www.ncbi.nlm.nih.gov/books/NBK2510/.

Casida JE, Durkin KA. (2015) Novel GABA receptor pesticide targets. Pesticide Biochem

Physiol. 121:22-30.

Chen L, Durkin KA, Casida J. (2006) Structural model for gamma-aminobutyric acid

receptor noncompetitive antagonist binding: widely diverse structures fit the same site.

Proc Natl Acad Sci USA. 103(13):5185-5190.

Cole LM, Casida JE. (1986) Polychlorocycloalkane insecticide-induced convulsions in

mice in relation to disruption of the GABA-regulated chloride ionophore. Life Sci. 39

(20):1855-62.

Collier ZA, Gust KA, Gonzalez-Morales B, Gong P, Wilbanks MS, Linkov I, Perkins EJ.

(2016) A weight of evidence assessment approach for adverse outcome pathways.

Regul Toxicol Pharmacol. 75:46-57.

Dichter MA, Ayala GF. (1987) Cellular mechanisms of epilepsy: A status report. Science

237: 157-164.

Durukan P, Ozdemir C, Coskun R, Ikizceli I, Esmaoglu A, Kurtoglu S, Guven M. (2009)

Experiences with endosulfan mass poisoning in rural areas. Eur J Emerg Med. 16(1):53-6.

Galanopoulou AS. (2008) GABAA Receptors in Normal Development and Seizures:

Friends or Foes? Curr Neuropharmacol. 6(1): 1–20.

Garcia-Reyero N, Habib T, Pirooznia M, Gust KA, Gong P, Warner C, Wilbanks M,

Perkins E. (2011) Conserved toxic responses across divergent phylogenetic lineages: a

meta-analysis of the neurotoxic effects of RDX among multiple species using

toxicogenomics. Ecotoxicology. 20(3):580-94.

http://www.ncbi.nlm.nih.gov/books/NBK2510/

18 │

Gong P, Hong H, Perkins EJ. (2015) Ionotropic GABA receptor antagonism-induced

adverse outcome pathways for potential neurotoxicity biomarkers. Biomarkers in Medicine

9(11):1225-39.

Hosie AM, Aronstein K, Sattelle DB, ffrench-Constant RH. (1997) Molecular biology of

insect neuronal GABA receptors. Trends Neurosci. 20(12): 578-583.

Ikeda T, Nagata K, Shono T, Narahashi T. (1998) Dieldrin and picrotoxinin modulation of

GABA(A) receptor single channels. Neuroreport 9(14):3189-3195.

Juarez EH, Ochoa-Cortes F, Miranda-Morales M, Espinosa-Luna R, Montano L M,

Barajas-Lopez C. (2013) Selectivity of antagonists for the Cys-loop native receptors for

ACh, 5-HT and GABA in guinea-pig myenteric neurons. Auton Autacoid Pharmacol, 34(1-

2):1-8.

Lomen-Hoerth C, Messing RO. (2010) Chapter 7: Nervous system disorders. In: Stephen

J. McPhee, and Gary D. Hammer (Eds), Pathophysiology of disease: an introduction to

clinical medicine (6th Edition). New York: McGraw-Hill Medical. ISBN 9780071621670.

McGonigle I, Lummis SC. (2010) Molecular characterization of agonists that bind to an

insect GABA receptor. Biochemistry. 49(13):2897-902.

Moon JM, Chun BJ, Lee SD. (2017) In-hospital outcomes and delayed neurologic sequelae

of seizure-related endosulfan poisoning. Seizure. 51:43-49.

Moses V, Peter JV. (2010) Acute intentional toxicity: endosulfan and other

organochlorines. Clin Toxicol (Phila). 48(6):539-44.

Newland CF, Cull-Candy SG. (1992) On the mechanism of action of picrotoxin on GABA

receptor channels in dissociated sympathetic neurones of the rat. J Physiol, 447: 191–213.

OECD. (2017) Users' Handbook Supplement to the Guidance Document for Developing

and Assessing AOPs. OECD Environment Directorate, Environment, Health and Safety

Division, Series on Testing & Assessment No. 233, Series on Adverse Outcome Pathways

No. 1, ENV/JM/MONO(2016)12, Paris, France.

Olsen RW. (2015) Allosteric ligands and their binding sites define γ-aminobutyric acid

(GABA) type A receptor subtypes. Adv Pharmacol. 73:167-202.

Olsen RW, Sieghart W. (2009) GABA A receptors: subtypes provide diversity of function

and pharmacology. Neuropharmacology. 56(1):141-8.

Parbhu B, Rodgers G, Sullivan JE. (2009) Death in a toddler following endosulfan

ingestion. Clin Toxicol (Phila). 47(9):899-901.

Raymond-Delpech V, Matsuda K, Sattelle BM, Rauh JJ, Sattelle DB. (2005) Ion channels:

molecular targets of neuroactive insecticides. Invert Neurosci, 5(3-4):119-133.

Roberts DM, Dissanayake W, Sheriff MHR, Eddleston M. (2004) Refractory status

epilepticus following self-poisoning with the organochlorine pesticide endosulfan. J

Clinical Neurosci. 11(7): 760-2.

Scharfman HE, Brooks-Kayal AR. (2014) Is Plasticity of GABAergic Mechanisms

Relevant to Epileptogenesis? In: Scharfman HE and Buckmaster PS (eds.), Issues in

Clinical Epileptology: A View from the Bench, Advances in Experimental Medicine and

Biology 813, pp.133-150.

Sieghart W. (1995) Structure and pharmacology of gamma-aminobutyric acid A receptor

subtypes. Pharmacol.Rev. 47(2):181-234.

https://aopwiki.org/wiki/index.php/Special:BookSources/9780071621670

│ 19

Stilwell GE, Saraswati S, Littleton JT, Chouinard SW. (2006) Development of a Drosophila

seizure model for in vivo high-throughput drug screening. Eur J Neurosci, 24(8):2211-22.

Taketo M , Yoshioka T (2000) Developmental change of GABA(A) receptor-mediated

current in rat hippocampus. Neuroscience 96(3):507-514.

Treiman DM. (2001) GABAergic mechanisms in epilepsy. Epilepsia, 42(Suppl. 3):8–12.

Williams LR, Aroniadou-Anderjaska V, Qashu F, Finne H, Pidoplichko V, Bannon D I et

al. (2011) RDX binds to the GABA(A) receptor-convulsant site and blocks GABA(A)

receptor-mediated currents in the amygdala: a mechanism for RDX-induced seizures.

Environ Health Perspect, 119(3):357-363.

20 │

Appendix 1

List of MIEs in this AOP

Event: 667: Binding at picrotoxin site, iGABAR chloride channel

Short Name: Binding at picrotoxin site, iGABAR chloride channel

Key Event Component

Process Object Action

GABA receptor

binding

gamma-aminobutyric acid receptor subunit

alpha-1 increased

GABA receptor

binding


alpha-5 increased

GABA receptor

binding


alpha-6 increased

AOPs Including This Key Event

AOP ID and Name Event Type

Aop:10 - Binding to the picrotoxin site of ionotropic

GABA receptors leading to epileptic seizures in adult brain

MolecularInitiatingEvent

Stressors

Name

Picrotoxin

Lindane

Fipronil

RDX

Alpha-endosulfan

Penicillin

Biological Context

Level of Biological Organization

Molecular

Cell term

Cell term

neuron


https://aopwiki.org/aops/10


│ 21

Organ term

Organ term

brain

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

Chemicals non-competitively bind at or near the central pore of the receptor complex (e.g.,

the picrotoxin site) and directly block chloride conductance through the ion channel

(Kalueff 2007). It has been postulated that they fit a single "big picrotoxin binding pocket"

in the chloride channel lumen lined by five TM2 segments (Kalueff 2007; Olsen 2006).

This hypothesis was examined with the β3 homopentamer by mutagenesis, pore structure

studies, ligand binding, and molecular modeling (Chen et al. 2006). Results suggest that

they fit the 2' to 9' pore region forming hydrogen bonds with the T6' hydroxyl and

hydrophobic interactions with A2', T6', and L9' alkyl substituents, thereby blocking the

channel. More computational evidence can be found in Sander et al. (2011), Carpenter et

al. (2013) and Zheng et al. (2014).






fruit fly Drosophila melanogaster High NCBI


dogs Canis lupus familiaris High NCBI


Life Stage Evidence

Adult High

Sex Applicability

Sex Evidence

Unspecific High

Theoretically, this MIE is applicable to any organisms that possess ionotropic GABA

receptors (iGABARs) in their central and/or peripheral nervous systems. Many reviews

(e.g., Hoisie et al. 1997; Buckingham et al. 2005; Michels and Moss 2007; Olsen and






22 │

Sieghart 2009) have summarized evidence of ubiquitous existence of iGABARs (GABAA-

R in vertebrates including the humans) in species spanning from invertebrates to human.

For instance, an ionotropic GABA receptor gene (GABA-receptor subunit-encoding Rdl

gene) was isolated from a naturally occurring dieldrin-resistant strain of D. melanogaster

(Ffrench-Constant et al., 1991,1993; Ffrench-Constant and Rocheleau, 1993). Nineteen

GABAA receptor genes have been identified in the human genome (Simon et al. 2004).

Direct evidence is mostly derived from in silico molecular modeling that docks ligands to

the binding pockets of iGABARs in human (Carpenter et al. 2013; Chen et al. 2006; Sander

et al. 2011), fruitfly and zebrafish (Zheng et al. 2014).

It should be noted that this MIE is limited to adult brains because of the age- or

developmental stage-dependent nature of iGABAR where GABA acts as an excitatory

(instead of inhibitory) neurotransmitter due to a higher intracellular chloride concentration

in immature or developing neurons (Taketo and Yoshioka 2000; Galanopoulou 2008; Ben-

Ari 2006).

This MIE is not applicable to GABAB receptors because, as metabotropic G-protein-

coupled receptors, they mediate slow and sustained inhibitory responses and are involved

in absence epilepsy (Han et al. 2012).

Key Event Description

Figure 1. Structure of ionotropic GABA receptors based on the consensus in multiple

literature reviews (Source: Gong et al. 2015).

Shown is a common subtype α1β2γ2 of GABAA receptors found in the mammalian CNS. (A) Five subunits

from three subunit subfamilies assemble to form a heteropentameric chloride permeable channel. (B)

Stoichiometry and subunit arrangement of the GABAA receptor. Also shown are the binding sites for GABA

and BZ. (C) Receptor subunits consist of four hydrophobic transmembrane domains (TM1-4), where TM2 is

believed to line the pore of the channel. The large extracellular N-terminus is the site for ligand binding as well

as the site of action of various drugs (e.g., picrotoxin and dieldrin). Each receptor subunit also contains a large

intracellular domain between TM3 and TM4, which is the site for various protein–protein interactions as well

as the site for post-translational modifications that modulate receptor activity. BZ: Benzodiazepines; CNS:

Central nervous system; TM: Transmembrane.

│ 23

As shown in Figure 1, binding of GABA opens up the chloride channel in the iGABAR

complex, leading to neuronal inhibition (Olsen et al. 2004). Non-competitive channel

blockers (e.g., fipronil, lindane, picrotoxin, penicillin and alpha-endosulfan) indirectly

modulate the iGABAR activity (i.e., alter the response of the receptor to agonist) by

noncompetitively binding at or near the central pore of the receptor complex (e.g., the

picrotoxin site), an allosteric site distinct from that of the orthosteric agonist binding site,

and inducing a conformational change within the receptor (Ernst et al. 2005; Johnston

2005).

Mounting evidence indicates that iGABAR chloride channel blockers bind to overlapping

but not identical sites (Kalueff 2007). Based on the mapping of convulsants' binding

domains to the iGABAR chloride ionophore (Olsen 2006), Kalueff (2007) proposed a "big

picrotoxin binding pocket" model for the ligand-binding area of ionophore. For

instance, bicyclophosphates, butyrolactones, pentylenetetrazole and penicillin act on the

N-terminus of TM2 (‘‘main’’ picrotoxin binding site), whereas picrotoxin may act on the

"main" as well as a hypothetical second ‘‘allosteric’’ picrotoxin site located on the C-

terminus of TM2 (Kalueff 2007; Olsen 2006). In spite of minor differences, these blockers

share similarity in channel blockage characteristics (e.g., voltage-dependency, binding to

closed channel, reversible, and non-competitive) and effects of channel state

(open/closed frequency and duration) (Kalueff 2007). Nevertheless, the 3D structure of

iGABAR ionophore is unavailable. More homology modeling work (similar to the electron

microscopic structure of nicotinic receptor channel (Unwin 2005)) may allow us to gain in-

depth understanding of the picrotoxin site's accessibility for and interactions with channel

blockers (i.e., convulsants), clarify the biological functions of the binding sites, and verify

the impact of mutations in TM2 on ligand binding activity (Kalueff 2007).

How it is Measured or Detected

Binding to a specific site on iGABAR can be determined using a variety of methods

including mutagenesis, pore structure studies, ligand binding, and molecular modeling

(more details on methods can be found in Chen et al. 2006). One should choose a method

in accordance with specific goal and also on the basis of available laboratory facilities. For

example, Atack et al. (2007) chose the radioligand [35S]t-butylbicyclophosphorothionate

(TBPS) binding assay to determine the binding properties (i.e., inhibition by TBPS,

picrotoxin, loreclezole and pentobarbital and modulation by GABA) at the convulsant

binding site.

References

Atack JR, Ohashi Y, McKernan RM. 2007. Characterization of [35S]t-

butylbicyclophosphorothionate ([35S]TBPS) binding to GABAA receptors in postmortem

human brain. Br J Pharmacol. 150(8):1066-74.

Buckingham SD, Biggin PC, Sattelle BM, Brown LA, Sattelle DB. 2005. Insect GABA

receptors: splicing, editing, and targeting by antiparasitics and insecticides. Mol Pharmacol

68(4):942-951.

Ben-Ari Y. 2006. Seizures beget seizures: the quest for GABA as a key player. Crit Rev

Neurobiol 18(1-2): 135-44.

24 │

Carpenter TS, Lau EY, Lightstone FC. 2013. Identification of a possible secondary

picrotoxin-binding site on the GABAA receptor. Chem Res Toxicol. 26(10):1444-54.

Chen L, Durkin KA, Casida JE. 2006. Structural model for gamma-aminobutyric acid


Proc Natl Acad Sci USA 103(13):5185-5190.

Ernst M, Bruckner S, Boresch S, Sieghart W. 2005. Comparative models of GABAA

receptor extracellular and transmembrane domains: important insights in pharmacology

and function. Mol Pharmacol 68(5):1291-1300.

Ffrench-Constant RH, Mortlock DP, Shaffer CD, MacIntyre RJ, Roush RT. 1991.

Molecular cloning and transformation of cyclodiene resistance in Drosophila: an

invertebrate gamma-aminobutyric acid subtype A receptor locus. Proc Natl Acad Sci USA

88:7209–7213.

Ffrench-Constant RH and Rocheleau TA. 1993 Drosophila gamma-aminobutyric acid

receptor gene Rdl shows extensive alternative splicing. J Neurochem 60:2323–2326.

Ffrench-Constant RH, Steichen JC, Rocheleau TA, Aronstein K, and Roush RT. 1993. A

single-amino acid substitution in a gamma-aminobutyric acid subtype A receptor locus is

associated with cyclodiene insecticide resistance in Drosophila populations. Proc Natl

Acad Sci USA 90:1957–1961.

Galanopoulou AS. 2008. GABAA Receptors in Normal Development and Seizures: Friends

or Foes? Curr Neuropharmacol. 6(1):1-20.

Gong P, Hong H, Perkins EJ. 2015. Ionotropic GABA receptor antagonism-induced

adverse outcome pathways for potential neurotoxicity biomarkers. Biomarkers in Medicine

9(11):1225-39.

Han HA, Cortez MA, Snead OC III. 2012. GABAB Receptor and Absence Epilepsy. In:

Noebels JL, Avoli M, Rogawski MA, et al., editors. Jasper's Basic Mechanisms of the

Epilepsies [Internet]. 4th edition. Bethesda (MD): National Center for Biotechnology

Information (US); 2012. Available from:

https://www.ncbi.nlm.nih.gov/books/NBK98192/.

Hosie AM, Aronstein K, Sattelle DB, Ffrench-Constant RH. 1997. Molecular biology of

insect neuronal GABA receptors. Trends Neurosci 20(12):578-583.

Johnston GA. 2005. GABA(A) receptor channel pharmacology. Curr Pharm Des

11(15):1867-1885.

Kalueff AV. 2007. Mapping convulsants' binding to the GABA-A receptor chloride

ionophore: a proposed model for channel binding sites. Neurochem Int 50(1): 61-68.

Michels G, Moss SJ. 2007. GABAA receptors: properties and trafficking. Crit Rev

Biochem Mol Biol 42(1):3-14.

Olsen RW. 2006. Picrotoxin-like channel blockers of GABAA receptors. Proc Natl Acad

Sci USA. 103:6081-82.

Olsen RW, Chang C-SS, Li G, Hanchar HJ, Wallner M. 2004. Fishing for allosteric sites

on GABAa receptors. Biochem Pharmacol. 68:1675-84.

Olsen RW, Sieghart W. 2009. GABAA receptors: subtypes provide diversity of function

and pharmacology. Neuropharmacology. 56(1):141-8.

│ 25

Sander T, Frolund B, Bruun AT, Ivanov I, McCammon JA, Balle T. 2011. New insights

into the GABAA receptor structure and orthosteric ligand binding: receptor modeling

guided by experimental data. Proteins. 79(5):1458-77.

Simon J, Wakimoto H, Fujita N, Lalande M, Barnard EA. 2004. Analysis of the set of

GABA(A) receptor genes in the human genome. J. Biol. Chem. 279(40), 41422–41435.

Taketo M , Yoshioka T. 2000. Developmental change of GABAA receptor-mediated

current in rat hippocampus. Neuroscience 96(3):507-514.

Unwin N. 2005. Refined structure of the nicotinic acetylcholine receptor at 4 A˚ resolution.

J Mol Biol. 346:967-89.

Zheng N, Cheng J, Zhang W, Li W, Shao X, Xu Z, Xu X, Li Z. 2014. Binding difference

of fipronil with GABAARs in fruitfly and zebrafish: insights from homology modeling,

docking, and molecular dynamics simulation studies. J Agric Food Chem 62(44):10646-

53.

26 │

List of Key Events in the AOP

Event: 64: Reduction, Ionotropic GABA receptor chloride channel conductance

Short Name: Reduction, Ionotropic GABA receptor chloride channel conductance

Key Event Component


GABA-gated chloride ion channel activity chloride decreased


AOP ID and Name Event

Type

Aop:10 - Binding to the picrotoxin site of ionotropic GABA receptors

leading to epileptic seizures in adult brain

KeyEvent

Biological Context


Cellular

Cell term

Cell term

neuron

Organ term

Organ term

brain




rats Rattus norvegicus High NCBI


Drosophila melanogaster Drosophila melanogaster High NCBI








│ 27

Banerjee et al. (1999) reported functional modulation of GABAA receptors by Zn2+,

pentobarbital, neuroactive steroid alphaxalone, and flunitrazepam in the cerebral cortex and

cerebellum of rats undergoing status epilepticus induced by pilocarpine.

Babot et al. (2007) measured the reduction in mouse GABAA receptor function by 3 μM

dieldrin using the GABA-induced 36Cl- uptake method.

Bromfield et al. (2006) reviewed evidence for GABAA receptors in human and mammalian

brains, whereas Narahashi (1996) and Costa (2015) reviewed organochlorine and some

pyrethroid compounds as insecticides with the target site of chloride channel.

Grolleau and Sattelle (2000) reported a complete blocking of inward current by 100 μM

picrotoxin in the wild-type RDL (iGABAR) of Drosophila melanogaster.


This key event occurs at the cellular level and is characterized by a dose-dependent post-

synaptic inhibition of membrane currents in iGABAR-containing cells, especially neuronal

cells (Dichter and Ayala 1987; Bromfield et al. 2006), leading to the reduction of iGABAR

chloride channel conductance.


The change in membrane conductance can be measured by determining the alteration (i.e.,

inhibition) in muscimol-stimulated (Banerjee et al. 1999) or GABA-induced uptake (Babot

et al. 2007) of 36Cl- in cortical and cerebellar membranes or primary cerebellar granule cell

cultures, prior to and after exposure to a GABA antagonist. Inglefield and Schwartz-Bloom

(1998) reported a Cl--sensitive fluorescent dye-based method where to measure real-time

changes in intracellular chloride concentration with UV laser scanning confocal

microscopy.

References

Babot Z, Vilaro MT, Sunol C. (2007) Long-term exposure to dieldrin reduces gamma-

aminobutyric acid type A and N-methyl-D-aspartate receptor function in primary cultures

of mouse cerebellar granule cells. J. Neurosci. Res. 85(16), 3687-3695.

Banerjee PK, Olsen RW, Snead OC, III. (1999) Zinc inhibition of gamma-aminobutyric

acid(A) receptor function is decreased in the cerebral cortex during pilocarpine-induced

status epilepticus. J Pharmacol Exp Ther 1999; 291(1):361-366.




http://www.ncbi.nlm.nih.gov/books/NBK2510

Costa LG. (2015) The neurotoxicity of organochlorine and pyrethroid pesticides. Handb

Clin Neurol. 131:135-48.

Dichter MA, Ayala GF. (1987) Cellular mechanisms of epilepsy: a status report. Science

237(4811), 157-164.

http://www.ncbi.nlm.nih.gov/books/NBK2510

28 │

Gong P. Hong HH, Perkins EJ. (2015) Ionotropic GABA receptor antagonism-induced

adverse outcome pathways for potential neurotoxicity biomarkers. Biomark. Med.

9(11):1225-39.

Grolleau F, Sattelle DB. (2000) Single channel analysis of the blocking actions of BIDN

and fipronil on a Drosophila melanogaster GABA receptor (RDL) stably expressed in a

Drosophila cell line. Br J Pharmacol. 130(8):1833-42.

Inglefield JR, Schwartz-Bloom RD. (1998) Optical imaging of hippocampal neurons with

a chloride-sensitive dye: early effects of in vitro ischemia. J Neurochem. 70(6):2500-9.

Narahashi T. (1996). Neuronal ion channels as the target sites of insecticides. Pharmacol

Toxicol. 79(1):1-14.

│ 29

Event: 669: Reduction, Neuronal synaptic inhibition

Short Name: Reduction, Neuronal synaptic inhibition

Key Event Component


chemical synaptic transmission decreased



Type



KeyEvent

Biological Context


Cellular

Cell term

Cell term

neuron





guinea pig Cavia porcellus High NCBI


Japanese quail Coturnix japonica High NCBI


Life Stage Evidence

Adult High








30 │

Sex Applicability

Sex Evidence

Unspecific High

See Juarez et al. (2013) for supporting evidence for Guinea pig. For rat, whole-cell in vitro

recordings in the rat basolateral amygdala (BLA) showed that RDX reduces the frequency

and amplitude of GABAA receptor mediated sIPSCs and the amplitude of GABA-evoked

postsynaptic currents, whereas in extracellular field recordings from the BLA, RDX

induced prolonged, seizure-like neuronal discharges (Williams et al, 2011).


A reduction in GABA-mediated inhibition of neuronal synaptic signaling is reflected as

decreased frequency and amplitude of iGABAR-mediated spontaneous inhibitory

postsynaptic currents (sIPSCs) or abolishment of GABA-induced firing action (Newland

and Cull-Candy 1992).


Juarez et al. (2013) used primary cultured neurons obtained from the guinea-pig small

intestine to detect picrotoxin concentration-dependent (and reversible) inhibition of

GABA-induced membrane currents. Williams et al. (2011) used whole-cell in vitro

recordings in the rat basolateral amygdala (BLA) to detect the reduced frequency and

amplitude of GABAA receptor mediated spontaneous inhibitory postsynaptic currents

(sIPSCs) and the amplitude of GABA-evoked postsynaptic currents, both of which were

induced by RDX.

References

Newland C F, Cull-Candy S G. On the mechanism of action of picrotoxin on GABA

receptor channels in dissociated sympathetic neurones of the rat. J Physiol 1992; 447: 191–

213.

Juarez E H, Ochoa-Cortes F, Miranda-Morales M, Espinosa-Luna R, Montano L M,

Barajas-Lopez C. Selectivity of antagonists for the Cys-loop native receptors for ACh, 5-

HT and GABA in guinea-pig myenteric neurons. Auton Autacoid Pharmacol 2013; 34(1-

2):1-8.

Williams L R, Aroniadou-Anderjaska V, Qashu F, Finne H, Pidoplichko V, Bannon D I et

al. RDX binds to the GABA(A) receptor-convulsant site and blocks GABA(A) receptor-

mediated currents in the amygdala: a mechanism for RDX-induced seizures. Environ

Health Perspect 2011; 119(3):357-363.

│ 31

Event: 682: Generation, Amplified excitatory postsynaptic potential (EPSP)

Short Name: Generation, Amplified excitatory postsynaptic potential (EPSP)

Key Event Component


excitatory postsynaptic potential occurrence



Type



KeyEvent

Biological Context


Cellular

Organ term

Organ term

brain








Life Stage Evidence

Adult High







32 │

Sex Applicability

Sex Evidence

Unspecific High

Miura et al. (1997) reported supporting evidence from guinea pigs whereas Dichter and

Ayala (1987) and Bromfield et al. (2006) summarized relevant studies on humans. Acker

et al. (2016) perform simultaneous two-photon voltage-sensitive dye recording with two-

photon glutamate uncaging in order to measure the characteristics (amplitude and duration)

of uncaging-evoked EPSPs in acute mouse brain slices.


In neuroscience, an excitatory postsynaptic potential (EPSP) is defined as a

neurotransmitter-induced postsynaptic potential change that depolarizes the cell, and hence

increases the likelihood of initiating a postsynaptic action potential (Purves et al. 2001). On

the contrary, an inhibitory postsynaptic potential (IPSP) decreases this likelihood. Whether

a postsynaptic response is an EPSP or an IPSP depends on the type of channel that is

coupled to the receptor, and on the concentration of permeant ions inside and outside the

cell. In fact, the only factor that distinguishes postsynaptic excitation from inhibition is the

reversal potential of the postsynaptic potential (PSP) in relation to the threshold voltage for

generating action potentials in the postsynaptic cell. When an active presynaptic cell

releases neurotransmitters into the synapse, some of them bind to receptors on the

postsynaptic cell. Many of these receptors contain an ion channel capable of passing

positively charged ions (e.g., Na+ or K+) or negatively charged ions (e.g., Cl-) either into or

out of the cell. In epileptogenesis, discharges reduced GABAA receptor-mediated

hyperpolarizing IPSPs by shifting their reversal potentials in a positive direction. At the

same time, the amplitudes of Schaffer collateral-evoked RS-α-amino-3-hydroxy-5-methyl-

4-isoxazolepropionic acid receptor-mediated EPSPs and action potential-independent

miniature EPSPs were enhanced, whereas N-methyl-d-aspartate receptor-mediated EPSPs

remained unchanged. Together, these changes in synaptic transmission produce a sustained

increase in hippocampal excitability (Lopantsev et al. 2009).


EPSPs are usually recorded by measuring electrical responses and changes in intracellular

calcium concentration using intracellular electrodes (Miura et al. 1997) or

recording extracellular electrical activity or potential using >20 electroencephalogram

(EEG) electrodes (often in clinical settings) (Bromfield et al. 2006). Recently, voltage-

sensitive dyes have been successfully used for measuring voltage responses from large

neuronal populations in acute brain slice preparations (Popovic et al. 2015; Acker et al.

2016).

References

Acker CD, Hoyos E, Loew LM. (2016) EPSPs Measured in Proximal Dendritic Spines of

Cortical Pyramidal Neurons. eNeuro. 3(2) ENEURO.0050-15.2016.

│ 33





Dichter MA, Ayala GF. (1987) Cellular mechanisms of epilepsy: A status report. Science

237:157-64.

Lopantsev V, Both M, Draguhn A. 2009. Rapid Plasticity at Inhibitory and Excitatory

Synapses in the Hippocampus Induced by Ictal Epileptiform Discharges. Eur J Neurosci

29(6):1153–64.

Miura M, Yoshioka M, Miyakawa H, Kato H, Ito KI. (1997) Properties of calcium spikes

revealed during GABAA receptor antagonism in hippocampal CA1 neurons from guinea

pigs. J Neurophysiol. 78(5):2269-79.

Popovic MA, Carnevale N, Rozsa B, Zecevic D. (2015) Electrical behaviour of dendritic

spines as revealed by voltage imaging. Nature Communications. 6:8436.

Purves D, Augustine GJ, Fitzpatrick D, Katz LC, LaMantia A-S, McNamara JO, Williams

SM (Eds). 2001. Neuroscience. 2nd edition. Chapter 7. Neurotransmitter Receptors and

Their Effects. Sunderland (MA): Sinauer Associates. Available from:




34 │

Event: 616: Occurrence, A paroxysmal depolarizing shift

Short Name: Occurrence, A paroxysmal depolarizing shift

Key Event Component


membrane depolarization occurrence



Type



KeyEvent

Biological Context


Tissue

Organ term

Organ term

brain







Life Stage Evidence

Adult High






│ 35

Sex Applicability

Sex Evidence

Unspecific High

Most of the supporting evidence comes from studies on human and rodents. See the reviews

of Bromfield (2006) and Lomen-Hoerth and Messing (2010) for examples.


A paroxysmal depolarizing shift (PDS) or depolarizing shift is a cellular manifestation of

epilepsy. As summarized by Lomen-Hoerth and Messing (2010), brain electrical activity is

nonsynchronous under normal conditions. In epileptic seizures, a large group of neurons

begin firing in an abnormal, excessive, and synchronized manner, which results in a wave

of depolarization known as a paroxysmal depolarizing shift (Somjen, 2004). Normally after

an excitatory neuron fires it becomes more resistant to firing for a period of time, owing in

part to the effect of inhibitory neurons, electrical changes within the excitatory neuron, and

the negative effects of adenosine. However, in epilepsy the resistance of excitatory neurons

to fire during this period is decreased, likely due to changes in ion channels or inhibitory

neurons not functioning properly. This then results in a specific area from which seizures

may develop, known as a "seizure focus".

Increased, abnormal neuron firing causes a wave of depolarization throughout the

brain/neuronal tissue. At the level of single neurons, epileptiform activity consists of

sustained neuronal depolarization resulting in a burst of action potentials, a plateau-like

depolarization associated with completion of the action potential burst, and then a rapid

repolarization followed by hyperpolarization. This sequence is also called paroxysmal

depolarizing shift (PDS). The bursting activity resulting from the relatively prolonged

depolarization of the neuronal membrane is due to influx of extracellular Ca2+, which leads

to the opening of voltage-dependent Na+ channels, influx of Na+, and generation of

repetitive action potentials. The subsequent hyperpolarizing afterpotential is mediated by

iGABA receptors and Cl- influx, or by K+ efflux, depending on the cell type (Bromfield et

al. 2006).


Paroxysmal depolarizing shifts can be measured in vitro using patch-clamp recording

technique (Kapur 2009) or micro-electrode arrays (Novellino et al. 2011) to determin

effects of chemicals on action potential patterns of neurons.

PDS can be detected in vivo using electroencephalography techniques (Niedermeyer and

da Silva 2005).

References

Bromfield EB, Cavazos JE, Sirven JI. 2006. An Introduction to Epilepsy [Internet]. West

Hartford (CT): American Epilepsy Society; Chapter 1, Basic Mechanisms Underlying

Seizures and Epilepsy. Available from: http://www.ncbi.nlm.nih.gov/books/NBK2510/.


36 │

Kapur J. 2009. GABA | Pathophysiology of Status Epilepticus. Encyclopedia of Basic

Epilepsy Research, pp 304-8.

Lomen-Hoerth C, Messing RO. 2010. Chapter 7: Nervous system disorders. Edited by

Stephen J. McPhee, and Gary D. Hammer, Pathophysiology of disease: an introduction to

clinical medicine (6th Edition). New York: McGraw-Hill Medical. ISBN 9780071621670.

Novellino A, Scelfo B, Palosaari T, Price A, Sobanski T, Shafer TJ, Johnstone AF, Gross

GW, Gramowski A, Schroeder O, Jügelt K, Chiappalone M, Benfenati F, Martinoia

S, Tedesco MT, Defranchi E, D'Angelo P, Whelan M. 2011. Development of micro-

electrode array based tests for neurotoxicity: assessment of interlaboratory reproducibility

with neuroactive chemicals. Front Neuroeng. 4:4.

Niedermeyer E, da Silva FL. 2005. Electroencephalography: basic principles, clinical

applications, and related fields. Lippincott Williams & Wilkins.

Somjen GG. 2004. Ions in the Brain Normal Function, Seizures, and Stroke. New York:

Oxford University Press. p. 167.

https://aopwiki.org/wiki/index.php/Special:BookSources/9780071621670

│ 37

List of Adverse Outcomes in this AOP

Event: 613: Occurrence, Epileptic seizure

Short Name: Occurrence, Epileptic seizure

Key Event Component


seizures occurrence


AOP ID and Name Event Type

Aop:10 - Binding to the picrotoxin site of ionotropic GABA

receptors leading to epileptic seizures in adult brain

AdverseOutcome

Biological Context


Individual







honeybee Apis mellifera High NCBI

eisenia fetida eisenia fetida High NCBI


Life Stage Evidence

Adult High









38 │

Sex Applicability

Sex Evidence

Unspecific High

Substance-induced epileptic seizures have been documented in a wide range of species

including invertebrates and vertebrates (see Tingle et al. (2003) and Gunasekara et al.

(2007) for reviews on the list of aquatic and terrestrial species affected by fipronil). For

instance, fipronil can induce seizures in fruit flies (Stilwell et al. 2006) and house flies (Gao

et al. 2007).


Blockage of the GABA-gated chloride channel reduces neuronal inhibition and induces

focal seizure. This may further lead to generalized seizure, convulsions and death

(Bloomquist 2003; De Deyn et al. 1990; Werner and Covenas 2011). For instance, exposure

to fipronil produces hyperexcitation at low doses and convulsion or tonic-clonic seizure

and seizure-related death at high doses (Gunasekara et al. 2007; Tingle et al. 2003; Jackson

et al. 2009).

As described in Bromfield et al. (2006), sizure propagation, the process by which a partial

seizure spreads within the brain, occurs when there is sufficient activation to recruit

surrounding neurons. This leads to a loss of surround inhibition and spread of seizure

activity into contiguous areas via local cortical connections, and to more distant areas via

long association pathways such as the corpus callosum. The propagation of bursting

activity is normally prevented by intact hyperpolarization and a region of surrounding

inhibition created by inhibitory neurons. With sufficient activation there is a recruitment of

surrounding neurons via a number of mechanisms. Of equal interest, but less well

understood, is the process by which seizures typically end, usually after seconds or minutes,

and what underlies the failure of this spontaneous seizure termination in the life-threatening

condition known as status epilepticus (Bromfield et al. 2006).


Electrophysiological measurements and physical (visual) observation (for mortality) are

the methods often used to detect epileptic seizure-related effects (Ulate-Campos et al.

2016). One may also visit http://www.mayoclinic.org/diseases-

conditions/epilepsy/diagnosis-treatment/diagnosis/dxc-20117234 for more information on

how medical doctors diagnose epilepsy in patients.

Recently, a new technique called micro-electrode array (MEA) recording has been

developed and tested both in vitro (Novellino et al. 2011) and ex vivo (Dossi et al. 2014).

MEAs, which are microfabricated devices embedding an array of spatially arranged

microelectrodes, provide a unique opportunity to simultaneously stimulate and record field

potentials, as well as action potentials of multiple neurons from different areas of the tissue

(Dossi et al. 2014). Thus, MEAs recordings constitute an excellent tool for studying the

spatio-temporal patterns of spontaneous interictal and evoked seizure-like events, the

mechanisms underlying seizure onset and propagation, and electrophysiological activity of

the neurons in response to chemical exposures (Novellino et al. 2011; Dossi et al. 2014).

http://www.mayoclinic.org/diseases-conditions/epilepsy/diagnosis-treatment/diagnosis/dxc-20117234

http://www.mayoclinic.org/diseases-conditions/epilepsy/diagnosis-treatment/diagnosis/dxc-20117234

│ 39

Regulatory Significance of the AO

As a neurotoxicity endpoint, information with regard to the seizure or epilepsy is often used

by regulators such as EPA, FDA and DHS for human and environmental health assessment

and regulation of chemicals, drugs and other materials. For instance, the Office of Pesticide

Programs (OPP) in US EPA, regulates, monitors and investigates the use of all pesticides

in accordance with the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA)

(https://www.epa.gov/laws-regulations/summary-federal-insecticide-fungicide-and-

rodenticide-act). Many pesticides like fipronil target the iGABAR causing seizure and

mortality. Another example is the regulatory actions of US FDA to ensure drug safety (see

https://www.fda.gov/Drugs/DrugSafety/ucm436494.htm).

References

Bloomquist JR. 2003. Chloride channels as tools for developing selective insecticides.

Arch. Insect Biochem. Physiol 54(4), 145-156.

Bromfield EB, Cavazos JE, Sirven JI, editors. 2006. An Introduction to Epilepsy [Internet].

West Hartford (CT): American Epilepsy Society. Chapter 1 Basic Mechanisms Underlying

Seizures and Epilepsy. Available from: http://www.ncbi.nlm.nih.gov/books/NBK2510/

De Deyn PP, Marescau B, Macdonald RL. 1990. Epilepsy and the GABA-hypothesis a

brief review and some examples. Acta Neurol. Belg. 90(2), 65-81.

Dossi E, Blauwblomme T, Nabbout R, Huberfeld G, Rouach N. 2014. Multi-electrode

array recordings of human epileptic postoperative cortical tissue.J Vis Exp. (92):e51870.

Gao JR, Kozaki T, Leichter CA, Rinkevich FD, Shono T, Scott JG. 2007. The A302S

mutation in Rdl that confers resistance to cyclodienes and limited crossresistance to fipronil

is undetectable in field populations of house flies from the USA. Pestic. Biochem. Physiol.

88, 66−70.

Gunasekara AS, Truong T, Goh KS, Spurlock F, Tjeerdema RS. 2007. Environmental fate

and toxicology of fipronil. J. Pestic. Sci. 32(3), 189-199.

Jackson D, Cornell CB, Luukinen B, Buhl K, Stone D. 2009. Fipronil Technical Fact Sheet.

National Pesticide Information Center, Oregon State University Extension Services,

Novellino A, Scelfo B, Palosaari T, Price A, Sobanski T, Shafer TJ, Johnstone AF, Gross

GW, Gramowski A, Schroeder O, Jügelt K, Chiappalone M, Benfenati F, Martinoia

S, Tedesco MT, Defranchi E, D'Angelo P, Whelan M. 2011. Development of micro-

electrode array based tests for neurotoxicity: assessment of interlaboratory reproducibility

with neuroactive chemicals.Front Neuroeng. 4:4.

Stilwell GE, Saraswati S, J. Troy Littleton JT, Chouinard SW. 2006. Development of a

Drosophila seizure model for in vivo high-throughput drug screening. European J Neurosci.

24, 2211-2222.

Tingle CC, Rother JA, Dewhurst CF, Lauer S, King WJ. 2003. Fipronil: environmental

fate, ecotoxicology, and human health concerns. Rev. Environ. Contam Toxicol. 176, 1-

66.

https://www.epa.gov/laws-regulations/summary-federal-insecticide-fungicide-and-rodenticide-act

https://www.epa.gov/laws-regulations/summary-federal-insecticide-fungicide-and-rodenticide-act

https://www.fda.gov/Drugs/DrugSafety/ucm436494.htm


40 │

Ulate-Campos A, Coughlin F, Gaínza-Lein M, Fernández IS, Pearl PL, Loddenkemper T.

2016. Automated seizure detection systems and their effectiveness for each type of seizure.

Seizure. 40:88-101.

Werner FM, Covenas R. 2011. Classical neurotransmitters and neuropeptides involved in

generalized epilepsy: a focus on antiepileptic drugs. Curr. Med. Chem. 18(32), 4933-4948.

│ 41

Appendix 2

List of Adjacent Key Event Relationships

Relationship: 666: Binding at picrotoxin site, iGABAR chloride channel leads to

Reduction, Ionotropic GABA receptor chloride channel conductance

AOPs Referencing Relationship

AOP Name Adjacency

Weight

of

Evidence

Quantitative

Understanding

Binding to

the

picrotoxin

site of

ionotropic

GABA

receptors

leading to

epileptic

seizures in

adult brain

adjacent High High

Evidence Supporting Applicability of this Relationship




zebrafish Danio rerio High NCBI


Drosophila melanogaster Drosophila melanogaster High NCBI

Due to the universal existence of iGABARs in the animal kingdom, it would be a very long

list of studies that provide supporting evidence with regard to taxonomic applicability of

this key event relationship. The following are two examples: Williams et al. (2011)

determined the binding affinity of RDX to the picrotoxin-binding site and the blockage of

GABA(A) receptor-mediated currents in the rat amygdala; Grolleau and Sattelle (2000)

reported a complete blocking of inward current by 100 μM picrotoxin in the wild-type RDL

(iGABAR) of Drosophila melanogaster.

Key Event Relationship Description

Acting as the major inhibitory neurotransmitter receptors, the ionotropic GABA receptors

(iGABARs) are ligand-gated ion channels (LGICs) (Carpenter et al. 2013). Upon binding

of an agonist (e.g., GABA), the iGABAR opens and increases the intraneuronal

concentration of chloride ions, thus hyperpolarizing the cell and inhibiting the transmission


















42 │

of the nerve action potential. iGABARs also contain many other modulatory binding

pockets that differ from the agonist-binding site. The picrotoxin-binding site is a

noncompetitive channel blocker site located at the cytoplasmic end of the transmembrane

channel (Olsen 2015). Binding to this pocket blocks GABA-induced chloride current,

hence reduces chloride conductance.

Evidence Supporting this KER

Biological Plausibility

The mechanisms for noncompetitive picrotoxin site binding-induced reduction in chloride

conductance have been investigated intensively for several decades. The consensus has

been reached with ample support of computational and experimental evidence.

Noncompetitive channel blockers fit the 2' to 9' pore region forming hydrogen bonds with

the T6' hydroxyl and hydrophobic interactions with A2', T6' and L9' alkyl substituents

(Chen et al. 2006), which is the primary binding site in the chloride channel lumen lined

by five TM2 segments, thereby blocking the channel. Recent evidence suggests that there

also exists a secondary modulatory pocket at the interface between the ligand-binding

domain and the transmembrane domain of the iGABAR (Carpenter et al. 2013). It is

believed that the two mechanisms mediate the blockage of chloride conductance (Yoon et

al. 1993; Carpenter et al. 2013).

Empirical Evidence

Numerous pharmacological and computational studies have lent strong support for this

relationship. For instance, picrotoxin, applied intracellularly, was capable of blocking

GABA-activated chloride current (Akaike et al. 1985). Recently, a computational study

using homology modeling, docking and molecular dynamics simulation methods revealed

that difference in binding affinity of fipronil with different iGABARs may lead to

differential toxicity (potency) (Zheng et al. 2014).

Uncertainties and Inconsistencies

As a heteropentameric receptor, the iGABAR consists of five protein subunits arranged

around a central pore that form an ion channel through the membrane. The subunits are

drawn from a pool of 19 distinct gene products, including six alpha, three beta, and three

gamma subunits. The high diversity of subunit genes, in combination with alternative

splicing and editing, leads to an enormous variety and, consequently, variability in function

and sensitivity. This constitutes the main source of uncertainties.

References

Akaike N. Hattori K, Oomura Y, Carpenter D0. 1985. Bicuculline and picrotoxin block γ-

aminobutyric acid-gated Cl-conductance by different mechanisms. Experientia 41:70-71.

Carpenter TS, Lau EY, Lightstone FC. 2013. Identification of a possible secondary

picrotoxin-binding site on the GABA(A) receptor. Chem Res Toxicol. 26(10):1444-54.

Chen L, Durkin KA, Casida J. 2006. Structural model for gamma-aminobutyric acid


Proc Natl Acad Sci USA, 103(13):5185-5190.

│ 43

Grolleau F, Sattelle DB. 2000. Single channel analysis of the blocking actions of BIDN

and fipronil on a Drosophila melanogaster GABA receptor (RDL) stably expressed in a

Drosophila cell line. Br J Pharmacol. 130(8):1833-42.

Olsen RW. 2015. Allosteric ligands and their binding sites define γ-aminobutyric acid

(GABA) type A receptor subtypes. Adv Pharmacol. 73:167-202.

Williams LR, Aroniadou-Anderjaska V, Qashu F, Finne H, Pidoplichko V, Bannon D I et

al. 2011. RDX binds to the GABA(A) receptor-convulsant site and blocks GABA(A)

receptor-mediated currents in the amygdala: a mechanism for RDX-induced seizures.

Environ Health Perspect. 119(3):357-363.

Yoon KW, Covey DF, Rothman SM. 1993. Multiple mechanisms of picrotoxin block of

GABA-induced currents in rat hippocampal neurons. J Physiol. 464:423-39.

Zheng N, Cheng J, Zhang W, Li W, Shao X, Xu Z, Xu X, Li Z. 2014. Binding difference

of fipronil with GABA(A)Rs in fruitfly and zebrafish: Insights from homology modeling,

docking, and molecular dynamics simulation studies. J Agric Food Chem. 62:10646-53.

44 │

Relationship: 667: Reduction, Ionotropic GABA receptor chloride channel

conductance leads to Reduction, Neuronal synaptic inhibition


AOP Name Adjacency

Weight

of

Evidence

Quantitative

Understanding

Binding to

the

picrotoxin

site of

ionotropic

GABA

receptors

leading to

epileptic

seizures in

adult brain

adjacent High High






iGABARs and synaptic neurons are present in animals throughout the animal kingdom,

therefore this event is applicable to a wide range of species from earthworm to humans.

This relationship has been shown directly in rats ( Williams et al. 2011) and guinea pig

(Juarez et al. 2013).


A decline in conductance through chloride channels in iGABARs causes a reduction in

GABA-mediated inhibition of neuronal synaptic signaling, which is reflected as decreased

frequency and amplitude of iGABAR-mediated spontaneous inhibitory postsynaptic

currents or abolishment of GABA-induced firing action (Newland and Cull-Candy 1992).

For instance, whole-cell in vitro recordings in the rat basolateral amygdala (BLA) showed

that RDX reduces the frequency and amplitude of GABAA receptor mediated spontaneous

inhibitory postsynaptic currents (sIPSCs) and the amplitude of GABA-evoked postsynaptic

currents, whereas in extracellular field recordings from the BLA, RDX induced prolonged,

seizure-like neuronal discharges (Williams et al. 2011). These pieces of cellular level

evidence support that binding to the GABAA receptor convulsant site is the primary

mechanism of seizure induction by RDX and that the key event of reduction of GABAergic
















│ 45

inhibitory transmission in the amygdala is involved in the generation of RDX-induced

seizures.



Chloride channels play an important role in regulating neuronal excitability, especially in

the context of fast synaptic inhibition mediated by GABAA receptors. But in order for

chloride channels to reduce excitability, chloride driving force must be maintained to keep

a dynamic balancing of chloride influx and efflux, which also involves a variety of other

ion species (Prescott 2014). If chloride regulation is compromised, the efficacy of fast

synaptic inhibition can be compromised with adverse effects such as reduced neuronal

inhibition.

Empirical Evidence

The GABAA receptor is part of a larger GABA/drug receptor-Cl− ion channel

macromolecular complex. An integral part of this complex is the Cl− channel. The binding

sites localized in or near the Cl− channel for GABA, benzodiazepines, barbiturates,

picrotoxin and anesthetic steroids modulate receptor response to GABA stimulation. The

GABA-binding site is directly responsible for opening the Cl− channel.

Electrophysiological studies of the GABAA-receptor complex indicate that it mediates an

increase in membrane conductance with an equilibrium potential near the resting level of

−70 mV. This conductance increase is often accompanied by a membrane

hyperpolarization, resulting in an increase in the firing threshold and, consequently, a

reduction in the probability of action potential initiation, causing neuronal inhibition (Olsen

and DeLorey 1999). This reduction in membrane resistance is accomplished by the GABA-

dependent facilitation of Cl− ion influx through a receptor-associated channel.

Channel blockers, such as the convulsant compound picrotoxin, cause a decrease in mean

channel open time. Picrotoxin acts on the gating process of the GABAA receptor channel

and works by preferentially shifting opening channels to the briefest open state (1 msec).

Experimental convulsants like pentylenetetrazol and the cage convulsant t-butyl

bicyclophosphorothionate (TBPS) act in a manner similar to picrotoxin, preventing Cl−

channel permeability (Macdonald and Olsen 1994).


As a heteropentameric receptor, the iGABAR consists of five protein subunits arranged

around a central pore to form an ion channel through the membrane. The subunits are drawn

from a pool of 19 distinct gene products, including six alpha, three beta, and three gamma

subunits. The high diversity of subunit genes, in combination with alternative splicing and

editing, leads to an enormous variety and, consequently, variability in function and

sensitivity. This constitutes the main source of uncertainties.

References

Juarez E H, Ochoa-Cortes F, Miranda-Morales M, Espinosa-Luna R, Montano L M,

Barajas-Lopez C. Selectivity of antagonists for the Cys-loop native receptors for ACh, 5-

HT and GABA in guinea-pig myenteric neurons. Auton Autacoid Pharmacol 2013; 34(1-

2):1-8.

46 │

Macdonald R L, Olsen R W. GABAA receptor channels. Annu. Rev. Neurosci.

1994;17:569–602.

Newland C F, Cull-Candy S G. On the mechanism of action of picrotoxin on GABA

receptor channels in dissociated sympathetic neurones of the rat. J Physiol 1992; 447: 191–

213.

Olsen R W, DeLorey T M. Chapter 16. GABA and Glycine: GABA Receptor Physiology

and Pharmacology. In: Siegel GJ, Agranoff BW, Albers RW, et al. (Eds), Basic

Neurochemistry: Molecular, Cellular and Medical Aspects (6th edition), Philadelphia:

Lippincott-Raven; 1999.

Prescott S A. Chloride channels. In: Jaeger D and Jung R (Eds.), Encyclopedia of

Computational Neuroscience, Springer, New York, 2014. pp.1-4.

Williams L R, Aroniadou-Anderjaska V, Qashu F, Finne H, Pidoplichko V, Bannon D I et

al. RDX binds to the GABA(A) receptor-convulsant site and blocks GABA(A) receptor-

mediated currents in the amygdala: a mechanism for RDX-induced seizures. Environ

Health Perspect 2011; 119(3):357-363.

│ 47

Relationship: 683: Reduction, Neuronal synaptic inhibition leads to Generation,

Amplified excitatory postsynaptic potential (EPSP)


AOP Name Adjacency

Weight

of

Evidence

Quantitative

Understanding

Binding to

the

picrotoxin

site of

ionotropic

GABA

receptors

leading to

epileptic

seizures in

adult brain

adjacent High Moderate






El-Hassar et al. (2007) and Li et al. (1999) provided evidence for rat whereas Bartolomei

et al. (2008) reported evidence for human.


GABAA receptors mediate two distinct forms of inhibition, phasic and tonic. The first

consists of fast inhibitory postsynaptic potentials (IPSPs), regulating point-to-point

communication between neurons. The second consists of a persistent inhibitory

conductance that plays a crucial role in regulating the membrane potential and network

excitability (Farrant and Nusser 2005). In the case of phasic inhibition, synaptic GABAA

receptors, facing presynaptic release sites, are activated by a brief exposure to a high

concentration of GABA released by exocytosis of presynaptic vesicles. Once released,

GABA diffuses throughout the neuropil before being taken up by selective plasma

membrane transporters, which contribute to the clearance of the neurotransmitter

(Cherubini and Conti 2001). In the case of tonic inhibition, extrasynaptic GABAA

receptors, localized away from the synapses, are persistently exposed to low concentration

of “ambient” GABA.

Fast inhibitory neurotransmission in the mammalian central nervous system (CNS) is

mediated primarily by the neurotransmitters GABA and glycine. Glycine is predominantly

used in the spinal cord and the brain stem, whereas GABA is more commonly used in the
















48 │

brain (Jentsch et al. 2002). As the dominant charge carrier through GABA-A receptors,

chloride is directly implicated in the efficacy of fast neuronal synaptic inhibition (Prescott

2014). The binding of GABA to GABAA receptors opens intrinsic anion channels, which

leads to a Cl− influx that hyperpolarizes the neuron and thereby inhibits postsynaptic

neuronal activity in the adult CNS (Jentsch et al. 2002). Neurons communicate through

action potentials along their axons, and those action potentials are electrical events that

depend on the movements of ions, particularly sodium and potassium, across the neuronal

cell membrane (Jefferys 2010). Postsynaptic conductance changes and the potential

changes that accompany them alter the probability that an action potential will be produced

in the postsynaptic cell. Postsynaptic potentials (PSPs) are called excitatory (or EPSPs) if

they increase the likelihood of a postsynaptic action potential occurring, and inhibitory (or

IPSPs) if they decrease this likelihood (Purves et al. 2001). Given that most neurons receive

inputs from both excitatory and inhibitory synapses, it is important to understand more

precisely the mechanisms that determine whether a particular synapse excites or inhibits its

postsynaptic partner. In order to generate large EPSPs underlying depolarization shift

(cause of interictal spike discharge or epileptic seizure), the normal small EPSPs must be

amplified (Dichter and Ayala 1987). Blocking of chloride channel by non-competitive

blockers at the picrotoxin convulsant site on GABAA receptors reduces IPSPs or increases

the probability of firing of the neuron, causing an enhancement of excitatory postsynaptic

action potentials (EPSPs) (Dichter and Ayala 1987).



Seizure often involves the reorganizations occurring around the synapse that are extremely

diverse and complex. What could be the functional consequences of synaptic

reorganization? The hypotheses are admittedly very speculative, since we do not know the

role of each parameter under physiological conditions. Very importantly, a drastic

alteration of one parameter (e.g., the loss or reduction of GABAergic inhibition, or its

transformation into excitation) may be without any functional impact (Bernard 2012). This

is a key concept derived from the work of Prinz et al. (2004) performed in the

stomatogastric system of the lobster, which has led to the concept that there are multiple

solutions to a given biological problem. The stomatogastric system, which generates a

rhythm vital for the animal, is composed of three nuclei connected to each other via

different neurotransmitter systems. Knowing the types of channels expressed by the

neurons in each nuclei and the type of connections, the researchers built a computer model

in which each parameter (amplitude of the ionic current, strength of the connection) could

take any biologically realistic value. They varied all the parameters, and selected the sets

of parameters that produced the same rhythm recorded in vivo. They found that there are

countless possible solutions, which produce the same behavior at the network level.

Importantly, they also found that the system is “resistant” even if one type of channel is not

expressed, or if a connection between two nuclei is missing. Further, the values taken by a

given parameter (among the sets of solutions) match the biological variability (Schulz et

al. 2007; Marder and Goaillard 2006). That is, the variability of a given parameter measured

in a biological system (e.g. amplitude of GABAA receptor-mediated currents) may just

reflect the different solutions that enable networks to function adequately. One might

therefore consider that all the modifications occurring in epileptic networks may simply

constitute the expression of another set of “solutions” to perform normal physiological

function. Seizures are, after all, very infrequent events, which suggests that most of the

│ 49

time the system can cope with various parameters permutations without engaging in

abnormal activity.

Nevertheless, important functional changes in epilepsy appear to stem from some of the

synaptic modifications identified so far, including interictal activity (Bernard 2012). A shift

to a depolarizing action in a minority of cells may be sufficient to favor the occurrence of

interictal spikes (Cohen et al. 2002). Interictal-like activity appears very early after the

initial insult in experimental models, and precedes (by days) and even predicts the

appearance of the chronic phase of epilepsy defined by recurrent seizures (El-Hassar et al.

2007; Williams et al. 2009; White et al. 2010). Using a crude model of hippocampal

circuitry, El-Hassar et al. (2007) have tried to determine the conditions sufficient for the

genesis of interictal spikes. Many different solutions exist, which include decreased

dendritic GABAergic inhibition and increased glutamatergic excitation (El-Hassar et al.

2007), in a range of values found experimentally (El-Hassar et al. 2007). This model does

not explain why interictal activity is not permanent in vivo, but suggests clues regarding its

underlying mechanisms. Since interictal activity is rarely encountered in non-epileptic

individuals, it has been proposed that it is pathological. Studies performed in vitro suggest

that interictal-like activity can produce long-term potentiation of synapses, thus

contributing to the construction of hyperexcitable networks (Dzhala and Staley 2003). The

presence of interictal-like activity during the earliest stages of epileptogenesis may not only

constitute biomarkers for at-risk patients, but also one core mechanism of epileptogenesis

(El-Hassar et al. 2007; Williams et al. 2007; White et al. 2010). One study performed in

patients with temporal lobe epilepsy suggests that the size of the epileptogenic zone

increases with the duration of epilepsy (Bartolomei et al. 2008). The brain regions outside

the epileptogenic zone (i.e. the irritative zone) are often characterized by the presence of

interictal spikes. Some of these regions become part of the epileptogenic zone as epilepsy

evolves in time (Bartolomei et al. 2008). It is therefore tempting to propose that interictal

spikes participate in the transformation of the irritative regions into epileptic ones.

Empirical Evidence

There are numerous studies that reported the linkage between inhibitory postsynaptic

potential (IPSP) reductions and enhanced excitatory postsynaptic potentials (EPSPs). For

instance, Li et al. (1999) examined synaptic potentials of neurons in inferior colliculus (IC)

cortex slice and the roles of GABA and glutamate receptors in generating these potentials.

Using a GABAA antagonist, they blocked IPSPs and paired pulse inhibition of EPSPs

(leading to amplified EPSPs) in the electrical stimulation of the IC commissure that elicited

only IPSPs (10% of cells), only EPSPs (51%), or both (38%).


A synapse involves three compartments: the presynaptic terminal, the postsynaptic site, and

the glial cell processes surrounding them. Many features of the synapse can be modified.

The number of synapses established by a given neuron on its targets can decrease

(“pruning” or death of the presynaptic neuron) or increase (sprouting, neosynaptogenesis).

The properties of the presynaptic terminal can be changed (release probability,

neurotransmitter concentration in vesicles, control by presynaptic receptors). On the

postsynaptic site, the number, subunit composition, and function (e.g., phosphorylation,

anchoring) of the receptors can be changed. Finally, alterations at the glial cell level may

affect the environment of the synapse and its function (neurotransmitter uptake, energy

supply to neurons, etc.). See Bernard (2012) for a detailed review on the state of our current

knowledge regarding the time-dependent reorganizations of GABAergic circuits at the

50 │

synaptic level during epileptogenesis and a discussion on the possible functional

consequences of these alterations (particularly on the fate of GABAergic circuits).

With respect to the cause of EPSP amplification, there exist at least four mechanisms other

than the withdrawal or reduction of inhibition in the epileptic foci of normal CNS (Dichter

and Ayala 1987): (i) frequency potentiation of EPSPs, (ii) changes in the space constant of

the dendrites (or spines) of the postsynaptic neuron, (iii) activation of the NMDA receptor

as the cell depolarizes as a result of a reduction in voltage-dependent block of the receptor

by Mg2+, and (iv) potentiation by neuromodulators that are released during the ID (for

example, norepinephrine, somatostatin, and acetylcholine). In addition to direct increases

in excitatory synaptic efficacy, the depolarizing effects of EPSPs can be supplemented by

several voltage-dependent intrinsic currents that exist in CNS neurons. These include

slowly inactivating Na+ and Ca2+ currents and a large, transient Ca2+ current that is likely

to be responsible for Ca2+-dependent action potentials.

The above synaptic modifications and diverse causes of EPSP amplification constitute the

uncertainty factors for the relationship between reduced IPSPs and amplification of EPSPs

at the affected GABAergic neurons.

References

Bartolomei F, Chauvel P, Wendling F. 2008. Epileptogenicity of brain structures in human

temporal lobe epilepsy: a quantified study from intracerebral EEG. Brain. 131:1818–30.

Bernard C. 2012. Alterations in synaptic function in epilepsy. In: Noebels JL, Avoli M,

Rogawski MA, et al. (Eds), Jasper's Basic Mechanisms of the Epilepsies [Internet]. 4th

edition. Bethesda (MD): National Center for Biotechnology Information (US); Website:


Cherubini E, Conti F. 2001. Generating diversity at GABAergic synapses. Trends

Neurosci. 24:155–162

Cohen I, Navarro V, Clemenceau S, Baulac M, Miles R. 2002. On the origin of interictal

activity in human temporal lobe epilepsy in vitro. Science. 298:1418–21.

Cossart R, et al. 2001. Dendritic but not somatic GABAergic inhibition is decreased in

experimental epilepsy. Nat Neurosci. 4:52–62.

Dichter MA, Ayala GF. 1987. Cellular mechanisms of epilepsy: A status report. Science

237:157-64.

Dzhala VI, Staley KJ. 2003. Transition from interictal to ictal activity in limbic networks

in vitro. J Neurosci. 23:7873–80.

El-Hassar L, et al. 2007. Cell domain-dependent changes in the glutamatergic and

GABAergic drives during epileptogenesis in the rat CA1 region. J Physiol. 578:193–211.

Farrant M, Nusser Z. 2005. Variations on an inhibitory theme: phasic and tonic activation

of GABA(A) receptors. Nat Rev Neurosci. 6:215–29.

Jefferys JGR. 2010. Advances in understanding basic mechanisms of epilepsy and seizures.

Seizure 19:638–46.

Jentsch TJ, Stein V, Weinreich F, Zdebik AA. 2002 Molecular Structure and Physiological

Function of Chloride Channels. Physiol Rev. 82(2):503-68.


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Li Y, Evans MS, Faingold CL. 1999. Synaptic response patterns of neurons in the cortex

of rat inferior colliculus. Hear Res. 137(1-2):15-28.

Marder E, Goaillard JM. 2006. Variability, compensation and homeostasis in neuron and

network function. Nat Rev Neurosci. 7:563–74.

Prescott SA. 2014. Chloride channels. In: Jaeger D and Jung R (Eds.), Encyclopedia of

Computational Neuroscience, Springer, New York, 2014. pp.1-4.

Prinz AA, Bucher D, Marder E. 2004. Similar network activity from disparate circuit

parameters. Nat Neurosci. 7:1345–52.

Purves D, Augustine GJ, Fitzpatrick D, et al. (Eds). 2001. Chapter 7. Neurotransmitter

Receptors and Their Effects. In: Neuroscience (2nd edition). Sunderland (MA): Sinauer

Associates. Website: http://www.ncbi.nlm.nih.gov/books/NBK10799/

Schulz DJ, Goaillard JM, Marder EE. 2007. Quantitative expression profiling of identified

neurons reveals cell-specific constraints on highly variable levels of gene expression. Proc

Natl Acad Sci USA. 104:13187–91.

White A, et al. 2010. EEG spike activity precedes epilepsy after kainate-induced status

epilepticus. Epilepsia. 51:371–83.

Williams PA, et al. 2009. Development of spontaneous recurrent seizures after kainate-

induced status epilepticus. J Neurosci. 29:2103–12.


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Relationship: 684: Generation, Amplified excitatory postsynaptic potential (EPSP)

leads to Occurrence, A paroxysmal depolarizing shift


AOP Name Adjacency Weight

of

Evidence

Quantitative

Understanding

Binding to

the

picrotoxin

site of

ionotropic

GABA

receptors

leading to

epileptic

seizures in

adult brain

adjacent Moderate Moderate






Numerous studies have documented experimental evidence in support of this relationship

even though the underlying mechanisms are still not completely understood. See reviews

of Bromfield et al. (2006) and Dichter and Ayala (1987) for studies using rat or human

tissues or cell lines as the experimental subject.


Blockage of the ion channel of the iGABAR causes membrane depolarization and a

reduction in inhibitory postsynaptic currents. This leads to the increased, abnormal neuron

firing that causes a wave of depolarization throughout the brain/neuronal tissue. At the level

of single neurons, epileptiform activity consists of sustained neuronal depolarization

resulting in a burst of action potentials, a plateau-like depolarization associated with

completion of the action potential burst, and then a rapid repolarization followed by

hyperpolarization. This sequence is called the paroxysmal depolarizing shift. The bursting

activity resulting from the relatively prolonged depolarization of the neuronal membrane is

due to influx of extracellular Ca2+, which leads to the opening of voltage-dependent Na+

channels, influx of Na+, and generation of repetitive action potentials. The subsequent

hyperpolarizing afterpotential is mediated by iGABA receptors and Cl- influx, or by K+

efflux, depending on the cell type (Bromfield et al 2006).
















│ 53



It has been proposed that as the potentiated EPSP begins to depolarize the neuron, a

threshold is reached for the development of a slowly inactivating Na+ current that amplifies

the depolarization. As depolarization continues, the low threshold Ca2+ current may turn on

to further depolarize the neuron, while NMDA-mediated excitatory synapses become more

effective. Eventually, both higher threshold Na+ and Ca2+ currents are activated, and the

neuron discharges with a burst of action potentials and an additional slow depolarization

(Herron et al. 1985; Dingledine et al. 1986). This hypothesis involves the interplay of both

synaptic and voltage-dependent intrinsic events that occur in normal central neurons.

An alternative hypothesis for PDS generation focuses more on changes in the intrinsic

properties of neurons resulting in the development of burst firing independent of a primary

change in synaptic interactions (Dichter and Ayala 1987).

Empirical Evidence

For the first hypothesis, Higashida and Brown (1986) and Madison et al. (1986) have

demonstrated that epilepsy occurs when the usual balance of these normal events is altered

by a change in synaptic efficacy or a change in the control of intrinsic membrane currents.

The reason that any given form of epilepsy may develop in a given brain region may depend

on (i) differences in densities and locations of channels on various neurons, (ii) the

interaction of intrinsic currents with one another and with synaptic currents under

physiological conditions, (iii) the local synaptic organization of a given area, and (iv) the

liberation of endogenous synaptic modulators that may alter the various voltage-dependent

membrane currents through second messenger pathways.

For the alternative hypothesis, many studies have shown that in epilepsy models, this is

most readily accomplished by inhibiting K+ currents and by allowing the slower Ca2+

currents to be expressed (see review by Dichter and Ayala (1987)). Under more "natural"

circumstances, a variety of possible mechanisms may contribute to the development of

endogenous burst propensity in the absence of exogenous epileptogenic agents: (i)

neuromodulators (acetylcholine, norepinephrine, and peptides) can reduce K+ currents

(although stimulation of endogenous pathways, even intensely, has not been shown to

produce sufficient inhibition of K+ currents to result in burst firing); (ii) both elevation of

extracellular K ([K+]) and reduction of extracellular Ca ([Ca2+]) can change membrane

characteristics and induce burst firing modes; and (iii) anatomical distortion and

redistribution of channels after injury and partial denervation as seen in chronic epileptic

foci may induce burst firing.


In addition to the above two hypotheses with empirical evidence, some investigators have

proposed that neurons with endogenous bursting characteristics must act as a pacemaker in

order for epileptiform activity to develop (see review by Dichter and Ayala (1987)). Such

neurons would be the CA2 and CA3 pyramidal cells in the hippocampus, layer IV and

superficial layer V neocortical pyramidal cells, or the abnormally burst-firing neurons in

chronic neocortical foci. This hypothesis is supported by the demonstration of the lower

threshold for the induction of interictal discharges by epileptogenic agents in CA2 and CA3

and layer IV, the spread of abnormal activity from these areas to nearby areas in some

54 │

experimental foci, and by the correlation of the number of bursting cells with the seizure

frequency in chronic foci.

However, this hypothesis has been challenged on theoretical grounds by models that

demonstrate that a system with either positive or negative feedback elements does not

require unstable individual elements in order to develop oscillating behavior. There is also

experimental evidence against the obligatory involvement of neurons with endogenous

burst-firing characteristics. Studies of in vivo hippocampal penicillin epilepsy and in vitro

low Ca2+-high K+ models of epilepsy indicate that area CAl is able to develop spontaneous

IDs and seizures independent of areas CA2 and CA3. In addition, neocortical and spinal

cord cultures, in which individual neurons do not discharge with intrinsic bursts, become

organized into small synaptic networks that show synchronized "burst" behavior-all as a

result of synaptic interactions. Thus it appears that endogenous, Ca2+-dependent bursts are

not strictly necessary for the development of synchronous bursting activity in a neural

network, although their presence may be facilitatory and CNS regions containing such

burst-firing neurons may have a particularly high epileptiform potential.

References

Bromfield EB, Cavazos JE, Sirven JI. 2006. Chapter 1, Basic Mechanisms Underlying





237: 157-164.

Dingledine R, Hynes MA, King GL. 1986. Involvement of N-methyl-D-aspartate receptors

in epileptiform bursting in the rat hippocampal slice. J Physiol. 380:175-89.

Herron CE, Williamson R, Collingridge GL. 1985. A selective N-methyl-D-aspartate

antagonist depresses epileptiform activity in rat hippocampal slices. Neurosci Lett.

61(3):255-60.

Higashida H, Brown DA. 1986. Two polyphosphatidylinositide metabolites control two

K+ currents in a neuronal cell. Nature. 323(6086):333-5.

Madison DV, Malenka RC, Nicoll RA. 1986. Phorbol esters block a voltage-sensitive

chloride current in hippocampal pyramidal cells. Nature. 321(6071):695-7.


│ 55

Relationship: 630: Occurrence, A paroxysmal depolarizing shift leads to Occurrence,

Epileptic seizure


AOP Name Adjacency

Weight

of

Evidence

Quantitative

Understanding

Binding to

the

picrotoxin

site of

ionotropic

GABA

receptors

leading to

epileptic

seizures in

adult brain

adjacent High Moderate






Most lines of supporting evidence come from studies using human and rodent epilepsy

models. See Dichter and Ayala (1987) and Jefferys (2010) for examples.


Dichter and Ayala (1987) reviewed our current understanding of the simple focal seizure

models, where interictal discharge (ID) and seizures seem most closely related. In acute

focal epilepsy, during the ID, thousands of neurons in the focus synchronously undergo an

unusually large depolarization (the paroxysmal depolarizing shift or PDS), superimposed

on which is a burst of action potentials. The PDS is followed by a hyperpolarizing potential

(the post-PDS HP) and neuronal inhibition. In areas surrounding the focus, many neurons

are inhibited during the ID. In distant projection areas, neurons can be excited briefly but

more often are inhibited during the ID, according to their synaptic interactions. Axons that

end within the focus generate action potentials, which can "backfire" and propagate

antidromically. In addition, during the ID, at the site of the focus, extracellular levels of K+

increase and levels of Ca2+ decrease, presumably because of exit of K+ from and entry of

Ca2+ into neuronal processes during the intense neuronal activity.
















56 │



As reviewed by Dichter and Ayala (1987), when seizures develop, at least in the acute

focus, the neurons show a characteristic sequence of events: the post-PDS HP becomes

smaller, gradually disappears, and is replaced by a depolarization, on top of which are

smaller depolarizing waves that resemble small PDSs. This series of events occurs

synchronously in the population of neurons within the focus, and the EEG develops after

discharges (ADs) after several successive IDs. The ADs become longer with each ID and

then progress into a seizure. Meanwhile, near and distant areas of brain are brought into the

seizure process, and the abnormal activity spreads. During this process, levels of

extracellular K+ continue to increase until they reach a steady-state level well above normal,

and levels of extracellular Ca2+ continue to decrease. Finally the seizure subsides, and the

neuronal membrane hyperpolarizes well beyond control level. It is not known whether this

orderly progression from IDs to seizures occurs in the same way in chronic epileptic foci

or in many forms of human epilepsy.

Empirical Evidence

As summarized by Dichter and Ayala (1987), the EEG hallmarks of focal epilepsy both in

animal models and in human epilepsy are the ictal, or seizure, discharge and the interictal

spike discharge (ID). The EEG spike most often represents an electrophysiological marker

for a hyperexcitable area of cortex and arises in or near an area with a high epileptogenic

potential. As such, it has been considered the earliest and simplest electrical manifestation

of the epileptic process and has been the target of extensive investigations. In some forms

of epilepsy, seizure discharges can be seen to originate electrically and anatomically from

the site of spike discharges, and the transition between spikes and seizures has been

analyzed in these simple models. In other forms of epilepsy, however, the exact relation

between spike discharge and the onset and localization of seizure discharges has been more

difficult to determine. Depth electroencephalography from humans with focal epilepsy has

demonstrated multiple patterns during the transition to seizure, only some of which

resemble that seen in acute experimental focal epilepsy. Whether these observations

indicate that the mechanisms underlying the transition in chronic human foci are different

from those of the simple acute model is not yet clear.


A crucial issue related to the development of the ID is how so many neurons within a focus

develop simultaneous depolarizations. Synchronization may occur by any of several

synaptic and nonsynaptic mechanisms: (i) recurrent synaptic excitation, (ii) antidromic

activation of the afferent fibers, (iii) ephaptic interactions due to large currents that flow

through extracellular spaces, (iv) changes in extracellular ionic concentrations, (v)

electrical coupling between cortical neurons, and (vi) the diffuse liberation of modulators

(Dichter and Ayala 1987). Two alternative hypotheses emerged, that could broadly be

categorized as the epileptic neuron versus the epileptic network. In practice it really is

impossible to divorce the two: epilepsy is essentially a collective phenomenon that requires

synchrony amongst large numbers of neurons, but the reason for the excessive synchrony

and excitation can be abnormal intrinsic properties (the epileptic neuron), or abnormal

circuitry (the epileptic network), or both (in most cases) (Jefferys 2010).

│ 57

References


237: 157-164.

Jefferys JGR. 2010. Advances in understanding basic mechanisms of epilepsy and seizures.

Seizure 19:638–46.

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